MGDF analogs

ABSTRACT

MGDF analogs having one or more changed glycosylation sites as compared to a naturally occurring MGDF sequence of a corresponding number of amino acids are disclosed. The invention also relates to DNA sequences encoding said MGDF analogs, recombinant plasmids and host cells for analog expression, and therapeutic compositions including such analogs.

This application is a continuation of application Ser. No. 08/388,779,filed Feb. 15, 1995 which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to MGDF analogs having at least onechanged O- or N-linked glycosylation site. The invention also relates toDNA sequences encoding these MGDF analogs, and recombinant plasmids andhost cells for analog expression.

BACKGROUND OF THE INVENTION

MGDF, or megakaryocyte growth and differentiation factor, is a recentlycloned cytokine that appears to be the major regulator of circulatingplatelet levels. See Bartley, T. D. et al., Cell 77:1117-1124 (1994);Lok, S. et al., Nature 369:565-568 (1994); de Sauvage, F. J. et al.,Nature 369:533-538 (1994); Miyazake, H. et al., Exp. Hematol. 22:838(1994); and Kuter, D. J. et al., PNAS USA, 91:11104-11108 (1994). MGDFis also referred to as thrombopoietin (TPO), mpl-ligand, andmegapoietin. Mature human MGDF is a protein having 332 amino acids intotal. The sequence of this protein and the corresponding cDNA are shownin FIG. 1 herein (SEQ. ID NOS.: 1 and 2).

Recombinant MGDF produced in both Chinese Hamster Ovary (CHO) and E.coli cells has been demonstrated to have a biological activity ofspecifically stimulating or increasing megakaryocytes and/or plateletsin vivo in mice, rats and monkeys. See e.g., Hunt, P. et al., Blood84(10):390A (1994). Human MGDF molecules that have been truncated sothat they extend at least 151 amino acids, starting from amino acidposition 1 in FIG. 1, retain biological activity in vivo. FIG. 2 (SEQ.ID NOS.: 3 and 4) shows one example of a truncated MGDF molecule having174 amino acids that has biological activity and was used to create MGDFanalogs in the examples section below. It is also possible to remove upto the first six amino acids at the N-terminus of the human sequenceMGDF protein and retain biological activity. Therefore, it appears thatbiological activity is retained within amino acids 7 to 151 (inclusive)of the mature amino acid sequence of human MGDF shown in FIG. 1.

In general, many cell surface and secretory proteins produced byeucaryotic cells are modified with one or more oligosaccharide groups.This modification, referred to as glycosylation, can dramatically affectthe physical properties of proteins and can also be important in proteinstability, secretion, and subcellular localization. Proper glycosylationcan be essential for biological activity. In fact, some genes fromeucaryotic organisms, when expressed in bacteria (e.g., E. coli) whichlack cellular processes for glycosylating proteins, yield proteins thatare recovered with little or no activity by virtue of their lack ofglycosylation.

Glycosylation occurs at specific locations or sites along thepolypeptide backbone and is usually of two types: O-linkedoligosaccharides are attached to serine (Ser) or threonine (Thr)residues while N-linked oligosaccharides (chains) are attached toasparagine residues when they are part of the sequence Asn-X-Ser/Thr,where X can be any amino acid except proline. X is preferably one of the20 naturally occurring amino acids other than proline. The structures ofN-linked and O-linked oligosaccharides and the sugar residues found ineach type are different. One type of sugar that is commonly found onboth is N-acetylneuraminic acid (hereafter referred to as sialic acid).Sialic acid is usually the terminal residue of both N-linked andO-linked oligosaccharides and, by virtue of its negative charge, mayconfer acidic properties to the glycoprotein.

As used herein glycosylation “sites” are amino acid residues that arestructurally able to link to glycosyl residues, although such sites mayor may not be actually linked to a glycosyl residue. As noted above,O-linked sites are either Ser or Thr residues, whereas N-linked sitesare either Asn-X-Ser or Asn-X-Thr, where X is defined as any amino acidother than Pro. Whether a given site is glycosylated with a glycosylchain is determined by the host cell in which the molecule is expressed,the amino acids neighboring the site, and other factors. As used herein,the number of “chains” attached to a given MGDF analog will be theaverage number of carbohydrate (i.e., glycosyl) chains attached to agiven MGDF molecule expressed by a particular host cell. Notably, theglycosylation sites for natural and corresponding recombinant MGDF willgenerally be the same, whereas the number of chains will possibly varydepending upon whether the particular host cell used for recombinantexpression attaches glycosyl chains to the same sites or not, ascompared to the natural source. Herein, whenever a comparison is madebetween recombinant and natural MGDF analogs, the same number of aminoacids will be compared, regardless of whether the natural sourceactually produces an MGDF molecule having that length. Thus, “natural”refers to the sequence employed in a particular species (such as human)rather than the length of the molecule actually expressed in suchnatural source.

Naturally occurring MGDF is a glycosylated molecule. The glycosylationpattern of natural MGDF is related to two key domains that have beenfound in MGDF. The sequence of the first approximately 151 amino acidsof human MGDF, corresponding to an active portion of the molecule, bearsnotable homology to erythropoietin (EPO), a cytokine capable ofstimulating production of erythrocytes, and is referred to as the“EPO-like” domain of human MGDF. The remaining amino acids of the matureprotein make up a so-called “N-linked carbohydrate” domain, since theyinclude most if not all of the sites for N-linked glycosylation. Inhuman MGDF, there are six N-linked glycosylation sites all contained inthe N-linked glycosylation domain. Both domains contain O-linkedglycosylation sites. There are an estimated 12-14 O-linked glycosylationchains in the molecule. Experimental evidence with human MGDF DNAexpressed recombinantly in CHO cells reveals that in the EPO-like domainat least two O-linked sites are glycosylated, at positions 1 (Ser) and37 (Thr).

Glycoproteins such as MGDF can be separated into different charged formsusing techniques such as isoelectric focusing (IEF). For example,several parties have reported IEF studies of crude and partiallypurified erythropoietin preparations (Lukowsky et al., J. Biochem.50:909 (1972); Shelton et al., Biochem. Med. 12:45 (1975); Fuhr et al.,Biochem. Biophys. Res. Comm. 98:930 (1981)).

In spite of the above information on glycosylation of MGDF molecules,there remains a need to obtain MGDF molecules having a differentglycosylation pattern and which retain or have improved biologicalactivity.

Accordingly, it is an object of the present invention to provide novelglycosylated MGDF molecules. It is a further object of this invention toprovide pharmaceutical compositions containing such molecules andmethods of treating conditions treatable by MGDF with the MGDF analogsof this invention.

SUMMARY OF THE INVENTION

In one embodiment, the subject invention relates to analogs of MGDFcomprising an amino acid sequence which includes at least one added, atleast one deleted, and/or a combination of at least one added anddeleted, site for glycosylation as compared to the corresponding naturalsequence MGDF. The added or deleted site(s) for glycosylation may resultin a greater or lesser number of carbohydrate chains, and higher orlower sialic acid content, than corresponding natural sequence MGDF,particularly human MGDF. For example, one type of analog could involvedeleting one or more N- or O-linked sites, and addition of one or moreN- or O-linked sites at the same or another position.

In another aspect of the above embodiment, the subject invention relatesto MGDF analogs comprising amino acid sequences which involvereplacement of one or more N- or O-linked glycosylation sites with oneor more non-naturally occurring sites. Thus, an N-linked site may bereplaced with a different N-linked site; an N-linked site may bereplaced with an O-linked site; an O-linked site may be replaced with adifferent O-linked site; and an O-linked site may be replaced with anN-linked site.

Combinations of any of the above changes are further encompassed withinthis invention.

The invention further encompasses DNA sequences encoding such MGDFanalogs, and recombinant plasmids and host cells for analog expression.

In all of the above cases, it is preferred that the change inglycosylation site result in a change in the number, location or type(N- vs. O-) of glycosyl chains in the resulting MGDF analog and retainsa biological activity of MGDF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA and amino acid sequence of human MGDF including asignal peptide (amino acids −21 to −1) and the mature amino acidsequence (1-332).

FIG. 2 shows the DNA and amino acid sequence of MGDF corresponding toamino acids 1-174 of the human mature MGDF sequence (SEQ ID NOS: 3 and4). The sequences flanking the coding regions have introduced XbaI andSalI cloning sites at the 5′ and 3′ ends respectively.

FIG. 3 shows a Western blot with E. coli and CHO MGDF. MK stands forMet-Lys, which is added to the N-terminus of MGDF for expression, andmay be cleaved off using a dipeptidase, such as cathepsin C. A moleculein which MK has been removed is referred to as desMK. Treatment with theglycosidases neuraminidase and O-glycanase is indicated.

FIG. 4 shows in vivo activity of E. coli and CHO MGDF in normal mice, interms of platelet counts. The data indicates that glycosylated MGDF (CHOmaterial) has superior activity than non-glycosylated (E. coli)material. This may be a result of increased half-life for theglycosylated material. For example, CHO 332 stands for human MGDF aminoacids 1-332 (FIG. 1) expressed in CHO cells.

FIG. 5 shows a Western blot analysis of COS cell supernatants ofrecombinant human MGDF and analogs 4, 6, 7, 9, 10, and 11. Theconstruction of the analogs is described in Example 4. Analogs 4, 7, 10have at least one additional carbohydrate chain as evidenced by slowergel mobility. The analog numbers correspond to analog numbers providedin Table 1 (e.g., 11 corresponds to analog N11). The control is N1 inTable 1.

FIG. 6 shows a Western blot analysis of COS cell supernatants ofrecombinant human MGDF and analogs 4, 5, 13, 14, and 15. Theconstruction of the analogs is described in Example 4. Analogs 4, 13,14, and 15 have at least one additional carbohydrate chain as evidencedby slower gel mobility.

FIG. 7 shows a Western blot analysis of COS cell supernatants of humansequence MGDF and indicated MGDF analogs after treatment withN-glycanase. The results indicate that the analogs have differentialglycosylation patterns.

FIG. 8 shows the results of a human megakaryocyte growth bioassay withMGDF analogs. Panels A and D are the positive and negative controlsrespectively. The well pictured in panel A received 37.5 pg of wild type(i.e., natural sequence) MGDF 1-174 COS-1 conditioned medium and showssubstantial megakaryocyte growth. Panel D received 1.5 ul of COS-1 mockconditioned medium and shows no growth. Panels B and C are MGDF 1-174analogs 7 and 10 respectively. Panel B received 9.0 pg of MGDF COS-1conditioned medium while panel C received 27 pg and both show excellentmegakaryocyte growth.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides MGDF with different glycosylation sitesas compared to natural MGDF having a corresponding sequence.

In a first embodiment, the subject invention relates to analogs of MGDFcomprising an amino acid sequence which includes at least one added, atleast one deleted, and/or at least one added and deleted, site forglycosylation as compared to corresponding natural sequence MGDF. Theadded or deleted site(s) for glycosylation may result in a greater orlesser number of carbohydrate chains, and higher or lower sialic acidcontent, than corresponding natural sequence MGDF, particularly humanMGDF. A combination of a deletion of one site and addition of anothersite would result in no net change in the number of sites, but rather, achange in location and/or type of site. Such combined change analogs arealso encompassed within this invention.

In a another aspect of the above embodiment, the subject inventionrelates to MGDF analogs comprising amino acid sequences which includereplacement of one or more N- or O-linked glycosylation sites with oneor more non-naturally occurring sites. Thus, an N-linked site may bereplaced with a different N-linked site; an N-linked site may bereplaced with an O-linked site; an O-linked site may be replaced with adifferent O-linked site; and/or an O-linked site may be replaced with anN-linked site.

The term “MGDF”, as used herein, includes naturally occurring MGDF,truncations of naturally occurring MGDF as well as non-naturallyoccurring polypeptides having an amino acid sequence and glycosylationsufficiently duplicative of that of naturally occurring MGDF to allowpossession of a biological activity of specifically stimulating growth,development and/or production of megakaryocytes and/or platelets.

In a preferred embodiment, MGDF is the product of the expression of anexogenous DNA sequence that has been transfected into a eukaryotic hostcell; that is, in a preferred embodiment the MGDF is “recombinant MGDF”.The preferred eucaryotic host is mammalian, particularly preferably CHOcells. Recombinant MGDF is advantageously produced according to theprocedures described herein and in the publications cited hereinregarding cloning and expression of MGDF.

Some additional preferred MGDF molecules have the following amino acidsequences, based on FIG. 1 herein:

MGDF 1-332 amino acids 1-332 of FIG. 1 MGDF 1-191 amino acids 1-191 ofFIG. 1 MGDF 1-183 amino acids 1-183 of FIG. 1 MGDF 1-174 amino acids1-174 of FIG. 1 MGDF 1-163 amino acids 1-163 of FIG. 1 MGDF 1-153 aminoacids 1-153 of FIG. 1 MGDF 1-152 amino acids 1-152 of FIG. 1 MGDF 1-151amino acids 1-151 of FIG. 1 MGDF 7-332 amino acids 7-332 of FIG. 1 MGDF7-191 amino acids 7-191 of FIG. 1 MGDF 7-183 amino acids 7-183 of FIG. 1MGDF 7-174 amino acids 7-174 of FIG. 1 MGDF 7-163 amino acids 7-163 ofFIG. 1 MGDF 7-153 amino acids 7-153 of FIG. 1 MGDF 7-152 amino acids7-152 of FIG. 1 MGDF 7-151 amino acids 7-151 of FIG. 1

It should be noted that MGDF 1-183, 1-191, 7-183, and 7-191 encompassone or two additional naturally-occurring glycosylation sites on theC-terminus thereof, as compared to shorter sequences. In each of theabove cases, Met-Lys may further be included in the N-terminus thereof.

The in vitro specific activities referred to herein are measurements ofrelative in vitro specific activities and are not measurements ofabsolute in vitro specific activities. For the purposes of thisapplication, the specific activities are used only to compare relativeactivities of MGDF analogs that have been assayed using the same assay,using the same conditions including the same internal standard, andhaving the same analysis of the data used to calculate specificactivity, etc.

As used herein the phrase “analog of MGDF” refers to MGDF with one ormore changes in the amino acid sequence of MGDF which result in a changein the type (N- or O-linked), number, or location of sites forcarbohydrate attachment. In a preferred embodiment, the change inglycosylation site(s) results in a change in the number of glycosylchains attached to the MGDF molecule. In a particularly preferredembodiment, the change in glycosylation site(s) adds at least one(generally 1-6, preferably 1-5, particularly preferably 1-3) glycosylchains, and most preferably the chain(s) is(are) added via N-linkage. Inanother particularly preferred embodiment, the MGDF analog retains atleast equivalent biological activity as compared to natural sequenceMGDF (e.g., human MGDF) and may possess substantially higher activity,as measured in assays for biological activity.

To prepare such analogs of MGDF, preferably they are generated bysite-directed mutagenesis resulting in additions, deletions, orsubstitutions of amino acid residues that add, eliminate or alter sitesthat are available for glycosylation. By “altered” is meant that a sitehas been deleted while another has been added at the same or anotherlocation as the deleted site. However, as is appreciated by thoseskilled in the art, other methods could result in a gene encoding sameamino acid sequence, and such methods are encompassed herein. Theresulting analogs may have fewer or more (preferably more) carbohydratechains than natural human/recombinant MGDF.

Addition of one or more carbohydrate (i.e., glycosyl) chains to MGDF isone important object of this invention. MGDF analogs having morecarbohydrate chains than those found in the correspondingnaturally-occurring amino acid sequence (e.g., 1-332 or 1-174, etc.) aregenerated by adding glycosylation sites which do not perturb thesecondary or tertiary conformation in a way that would substantiallyreduce biological activity. As used herein the “naturally-occurring”MGDF refers to an amino acid sequence having the corresponding number ofamino acids as the relevant analog, even if the particular length ofMGDF species is not actually expressed in the native species.Advantageously, the analog of MGDF has up to 6 additional sites forN-glycosylation or O-glycosylation, resulting in the addition of up to 6additional N-linked or O-linked carbohydrate chains.

For example, a Pro at position 30 is replaced by an Asn and a Val atposition 32 is replaced by a Thr to give the sequence Asn-Glu-Thr, whichserves as a new site for N-glycosylation (analog 4 below; see Table 1).Analogs may also be constructed which have two or more additionalN-linked chains by combining mutations; for example, analogs 4 and 10described in Table 1 may be combined to yield an analog with twoadditional sites for carbohydrate addition (i.e., analog N15 in Table1). In a like manner analogs with three or more added chains can beconstructed. As will be appreciated by those skilled in the art, thesubject invention includes many other analogs of MGDF having differentsites for glycosylation (in terms of number, type or location of site).The MGDF analogs of this invention are in all cases particularlypreferably based on MGDF having a human amino acid sequence (see FIGS. 1and 2); however, analogs based on MGDF sequences from other species(e.g., dog, pig, monkey, mouse or rat) are also contemplated herein.

Also included within the analogs of this invention are analogs whichhave one or more amino acids extending from the carboxy terminal end ofMGDF wherein the carboxy terminal extension provides at least oneadditional carbohydrate site. The carboxy terminus of MGDF will varydepending upon the particular form of MGDF used (e.g., MGDF 1-332 aminoacids, or MGDF 1-163 amino acids). An additional carbohydrate site maybe added to the carboxy terminus of an MGDF species by adding aminoacids to the carboxy terminus, such amino acids containing one or moreN- or O-linked glycosylation sites.

Table 1 lists some exemplary MGDF analogs which have additional sitesfor N-linked carbohydrate chains. The analogs have the sequenceAsn-X-Ser or Asn-X-Thr included at various positions in the human MGDFpolypeptide chain based on the human amino acid sequences to createN-linked sites. Table 1 also lists those analogs which add at least oneadditional N-linked carbohydrate chain, as evidenced by the migration ofthe glycoproteins on SDS gels (see, Example 6).

Also encompassed by the present invention are DNA sequences encoding theMGDF analogs disclosed herein, preferably those encoding analogs havingadditional sites for N-linked chains. Procedures used to introducechanges into the MGDF DNA sequence for the purpose of creating, deletingand/or altering attachment sites for carbohydrates are disclosed inExample 4.

These MGDF analogs can be the product of expression of an exogenous DNAsequence, i.e., produced through recombinant DNA technology, they can bechemically synthesized products or they may be produced by combinedmethods. An exogenous DNA sequence comprises cDNA, genomic DNA orchemically synthesized DNA encoding an MGDF analog. Recombinant DNAplasmids and eucaryotic host cells useful for the expression of saidanalogs are also provided. Expression vectors include any vector whichis capable of expressing cloned DNA sequences in a eucaryotic host cell,particularly those vectors used for expression in COS and CHO cells.Examples of such vectors include plasmids pDSRα and pDSRα2, see Mol.Cell. Biol. 8:466-472 (1988); WO 91/13160 (1991); and WO 90/14363(1990). The cultivation of COS and CHO host cells expressing MGDFanalogs was carried out using standard procedures known to those skilledin the art.

Changing the number, type or location of carbohydrate chains attached toMGDF may confer advantageous properties such as increased solubility,greater resistance to proteolysis, reduced immunogenicity, increasedserum half-life, and increased biological activity.

Conditioned media from COS cells expressing MGDF analogs N2-N15 (N1 ishuman MGDF 1-174; see FIG. 2) were analyzed for in vitro biologicalactivity and the results shown in Table 4.

Another embodiment of the invention relates to mammalian (e.g., ChineseHamster Ovary, CHO) host cells which preferentially synthesize MGDF oranalogs of MGDF having greater than a specific number of sialic acidsper molecule, e.g. greater than that found in MGDF 1-332, 1-174, 1-163,or 1-151 produced naturally or recombinantly in a eucaryotic cell.

The sialic acid content of the MGDF molecule may affect its in vivobiological activity. For example, tetraantennary (four-branched)N-linked oligosaccharides most commonly provide four possible sites forsialic acid attachment while bi- and triantennary oligosaccharides,which can substitute for the tetraantennary form at asparagine-linkedsites, commonly have at most only two or three sialic acids attached.O-linked oligosaccharides commonly provide two sites for sialic acidattachment. Thus, MGDF molecules with N-linked carbohydrate substitutedfor O-linked carbohydrate can accommodate two additional sialic acidsper chain provided the N-linked oligosaccharides are tetraantennary.Mammalian cell cultures are screened for those cells that preferentiallyadd tetraantennary chains to recombinant MGDF, thereby maximizing thenumber of sites for sialic acid attachment.

Dihydrofolate reductase (DHFR) deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins including recombinant MGDF.

Compositions comprising a therapeutically effective amount of an MGDFanalog in accordance with this together with a suitable diluent,adjuvant and/or carrier useful in MGDF therapy are further encompassedby this invention. A “therapeutically effective amount” as used hereinrefers to that amount which provides therapeutic effect for a givencondition and administration regimen.

The present compositions can be systemically administered parenterally.Alternatively, the compositions may be administered intravenously orsubcutaneously. When systemically administered, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, with due regardto pH, isotonicity, stability and the like, is within the skill of theart. The specific route chosen will depend upon the condition beingtreated. The administration of MGDF or MGDF analogs is preferably doneas part of a formulation containing a suitable carrier, such as humanserum albumin, a suitable diluent, such as a buffered saline solution,and/or a suitable adjuvant. The required dosage will be in amountssufficient to raise the platelet levels of patients and will varydepending upon the severity of the condition being treated, the methodof administration used and the like.

The conditions to be treated by the methods and compositions of thepresent invention are generally those which involve an existingmegakaryocyte/platelet deficiency or an expected megakaryocyte/plateletdeficiency in the future (e.g., because of planned surgery). Suchconditions will usually be the result of a deficiency (temporary orpermanent) of active MGDF in vivo. The generic term for plateletdeficiency is thrombocytopenia, and hence the methods and compositionsof the present invention are generally available for treatingthrombocytopenia.

Thrombocytopenia (platelet deficiencies) may be present for variousreasons, including chemotherapy and other therapy with a variety ofdrugs, radiation therapy, surgery, accidental blood loss, and otherspecific disease conditions. Exemplary specific disease conditions thatinvolve thrombocytopenia and may be treated in accordance with thisinvention are: aplastic anemia, idiopathic thrombocytopenia, metastatictumors which result in thrombocytopenia, systemic lupus erythematosus,splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic aciddeficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, andparoxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDSresult in thrombocytopenia (e.g., AZT). Certain wound healing disordersmight also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery, an MGDF analog of the present invention could be administeredseveral days to several hours prior to the need for platelets. Withregard to acute situations, e.g., accidental and massive blood loss, anMGDF analog could be administered along with blood or purifiedplatelets.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the daily regimen should be in the range of 0.01-1000 micrograms of MGDFanalog per kilogram of body weight.

The therapeutic methods, compositions and polypeptides of the presentinvention may also be employed, alone or in combination with othercytokines, soluble Mpl (i.e., MGDF) receptor, hematopoietic factors,interleukins, growth factors or antibodies in the treatment of diseasestates characterized by other symptoms as well as platelet deficiencies.It is anticipated that an MGDF analog molecule will prove useful intreating some forms of thrombocytopenia in combination with generalstimulators of hematopoiesis, such as IL-3 or GM-CSF. Othermegakaryocytic stimulatory factors, i.e., meg-CSF, stem cell factor(SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or othermolecules with megakaryocyte stimulating activity may also be employedwith MGDF. Additional exemplary cytokines or hematopoietic factors forsuch co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), IFN-beta, or IFN-gamma. It may further be useful toadminister, either simultaneously or sequentially, an effective amountof a soluble mammalian Mpl receptor, which appears to have an effect ofcausing megakaryocytes to fragment into platelets once themegakaryocytes have reached mature form. Thus, administration of MGDFanalog (to enhance the number of mature megakaryocytes) followed byadministration of the soluble Mpl receptor (to inactivate the analog andallow the mature megakaryocytes to produce platelets) is expected to bea particularly effective means of stimulating platelet production. Thedosage recited above would be adjusted to compensate for such additionalcomponents in the therapeutic composition. Progress of the treatedpatient can be monitored by conventional methods.

Additional modifications of the analogs of this invention may also becarried out, e.g., to increase activity, stability, half-life, etc. Forexample, pegylation (poly- or mono-) could be added to the MGDF analog.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.The MGDF standard used in the bioassays employed in the Examples is arecombinant MGDF standard that was expressed in E. coli, refolded intoan active conformation and purified. Thus, only relative specificactivities are being measured.

EXAMPLE 1 Construction of MGDF 1-174

Human MGDF gene encoding amino acids 1-174 of FIG. 2 was generated froma human fetal liver cDNA library (Bartley et al, Cell 77: 1117-1124(1994) by polymerase chain reaction (PCR). The 5′ PCR primer encoded theamino terminus of human MGDF, an XbaI site, and an optimized Kozaksequence. The 3′ primer contained a termination codon and a SalIrestriction site. The amplified DNA fragment was digested with XbaI andSalI then ligated to XbaI and SalI cut pDSRα2. The resultant plasmid,pDSRα2 MGDF 1-174 was used for mammalian cell expression. The sequenceof the resulting gene is shown in FIG. 2.

Plasmid DNA containing MGDF 1-174 was digested with XbaI and SalIrestriction enzymes, the resulting DNA fragments were subjected toagarose gel electrophoresis, and the 605 nt MGDF 1-174 DNA fragment wasisolated from the gel using a GeneClean™ kit and procedures supplied bythe manufacturer (BIO 101, Inc.). Plasmid pDSRα2 as described in WO90/14363 (1990) was also digested with XbaI and SalI restriction enzymesand the nt vector fragment was recovered. Ligation of the two fragmentsresults in pDSRα2 (MGDF 1-174).

EXAMPLE 2 Expression of MGDF 1-174 in CHO Cells and Purification

Dihydrofolate reductase deficient (DHFR⁻) Chinese Hamster Ovary (CHO)cells were transfected with pDSRα2-MGDF 1-174. A 100 mm tissue culturedish was plated with 1×10⁶ CHO DHFR⁻ cells grown in CHO D⁻ medium (DMEM,10% Fetal bovine serum, 1% penicillin/streptomycin/glutamine, 1%nonessential amino acids(Gibco) and 1% HT supplement (Gibco)) the daybefore transfection. Four transfections were performed. For eachtransfection, plasmid DNA (50 μg) was linearized by digesting with Pvu Iand Buffer H (Boehringer Mannheim). A DNA precipitate was then formedand added to the plates dropwise as per the Mammalian Cell TransfectionKit (Specialty Media). After 24 hours in a tissue culture incubator themedium was replaced with fresh CHO D− medium. Twenty four hours laterthe cells were split into 96 well tissue culture plates with 100 ul ofCHO select medium (D-MEM, 5% dialyzed fetal Bovine serum, 1%penicillin/streptomycin/glutamine, 1% nonessential amino acids (Gibco))per well and transformants were selected. Medium was changed weeklyuntil colonies appeared. After two weeks, MGDF expression was screenedfor using the 32D cell proliferation assay described below (see Example9). Those clones expressing in excess of 1×10⁵ units/ml were expandedand frozen in cryogenic storage. One clone was expanded for rollerbottle production and approximately 8 liters of conditioned medium wasproduced.

Plasmid pDSRα2 containing MGDF 1-174 cDNA was transfected intoDHFR-deficient CHO cells as explained above. Two liters of serum-freeCHO cell conditioned medium (5.0% D-MEM, 50% HAMS-F12, 1%penicillin/streptomycin/glutamine, 1% nonessential amino acids (Gibco))from roller bottles seeded with CHO cells expressing MGDF 1-174 wasconcentrated 15 fold using a 2 L Amicon Model 2000 stirred cell and a10,000 dalton molecular weight cut-off membrane (YM10, Amicon).Forty-five milliliters of concentrated conditioned medium was thenloaded directly onto a 4 ml hu-MPL-X affinity column at a flow rate of0.4 ml/min using a Pharmacia FPLC. The affinity column was constructedby coupling 1.5-2.5 milligrams of Mpl-X (the soluble extra-cellulardomain of the Mpl receptor) per milliliter of Pharmacia CNBR activatedSepharose resin as recommended by the manufacturer. After loading, thecolumn was washed with 16 ml of phosphate buffered saline (PBS; 10 mMNa.PO₄ pH 6.8/150 mM NaCl) and then 24 ml of 10 mM Tris, pH 8.0/1M NaCl.MGDF(1-174) was eluted with 40 ml of 20 mM CAPS (3-[Cyclohexylamino]-1propanesulfonic acid) pH 10.5/1M NaCl/5 mM CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) in 6 ml fractions. The secondfraction yielded a single band on a 14% SDS gel. This material wasconcentrated and buffer exchanged against a saline solution of 0.9% NaCland was biologically active in vitro and in vivo. Other forms of CHOcell expressed MGDF were purified in a similar manner.

EXAMPLE 3 In Vivo Biological Activity of rHuMGDF

Platelet counts from mice treated with various forms of r-HuMGDF weremeasured and the results are presented in FIG. 4. CHO-derived MGDF1-332, 1-174, 1-163, and 1-153 were produced and purified byMpl-receptor affinity chromatography. E. coli-derived Met-Lys-MGDF1-332, Met-Lys-MGDF 1-174, Met-Lys-MGDF 1-163 and Met-Lys-MGDF 1-153were produced and purified by conventional chromatography. The indicatedconcentration of each form was administered subcutaneously into normal,female Balb/c mice once daily for 5 days. Test bleeds from a smalllateral cut in a tail vein were collected 24 hours after the lastinjection. Blood cell analyses were performed with a Sysmex electronicblood cell analyser (Baxter Diagnostics, Inc. Irvine, Calif.). Data arerepresented as the mean of determinations of 4 animals, +/−standarderror of the mean. Other blood cell parameters such as total white bloodcell counts or red blood cell counts were not affected by thesetreatments (data not shown).

FIG. 4 shows platelet counts from mice treated with various forms of CHOcell-derived (solid lines) or E. coli-derived (dashed lines) recombinanthuman MGDF. Normal, female Balb/c mice were injected subcutaneously withthe indicated concentration of r-HuMGDF for 5 consecutive days. Twentyfours hours after the last injection, platelet counts were determinedwith an electronic cell counter. The results indicate that CHO cellexpressed forms of MGDF have an increased in vivo activity relative tothe same forms of MGDF produced in E. coli. As described in Example 6,the CHO cell expressed forms of MGDF all contain N and/or O-linkedcarbohydrate and the E. coli expressed MGDF forms do not. This indicatesthat the carbohydrate enhances the in vivo activity of MGDF. Theincreased in vivo activity conferred by the carbohydrate may be a resultof increased circulatory half life, increased stability or a combinationof both.

EXAMPLE 4 Construction of MGDF Analogs

Procedures for generating additional glycosylation sites for MGDF aredescribed below.

The following oligonucleotide primers were synthesized for use in invitro mutagenesis to prepare analogs N2-N14 (see Table 1 for thestructures of these analogs):

N2 CCCATGTCAATCACAGCAGACT SEQ ID NO.: 5 N3 CTTCACAGCAACCTGAGCCAGT SEQ IDNO.: 6 N4 CAGTGCAACGAGACCCACCCTTTG SEQ ID NO.: 7 N5GCCTACAAATGTCACGCTGCCTGCT SEQ ID NO.: 8 N6 CCCACTTGTAACTCATCCCTC SEQ IDNO.: 9 N7 CAACTGAACGCCACTTGTCTCTCA SEQ ID NO.: 10 N8ACTTGTCTCAACTCCACCCTGGGGGA SEQ ID NO.: 11 N9 CTCCTGGGGAACCTTTCTGGA SEQID NO.: 12 N10 GACCACAAATCACACCGATCCCAAT SEQ ID NO.: 13 N11ACCCTTTGTCTACAAATGTCACGCTGCCTGCT SEQ ID NO.: 14 N12TCTCTCAAACCTCACGGGGGAGCTT SEQ ID NO.: 15 N13 TGGAAAAATCAGACGGAGGAGAC SEQID NO.: 16 N14 TGGAGGAGAACAAGACACAGGACAT SEQ ID NO.: 17

To construct m13 mp18 MGDF 1-174, the gene of FIG. 2 was introduced intoXbaI and SalI restriction enzyme digested m13mp18 DNA. Single strandedDNA was recovered from supernatants of E. coli strain RZ1032 infected bym13mp18 (MGDF 1-174) as described by Kunkel et al., Methods in Enzymol.154:367 (1987) and Messing, Methods in Enzymol. 101:20 (1983). For invitro mutagenesis approximately 0.5 μg of single-stranded DNA and 0.125pmole of one of the synthetic primers described above were mixed with 6μl of buffer (250 mM Tris pH 7.8, 50 mM MgCl₂, 50 mM dithiothreitol and1% Bovine serum albumin (BSA-Pharmacia)). The primers were previouslykinased with ATP and T4 polynucleotide kinase prior to addition. Forannealing of the primer to the template, the reaction volume wasadjusted to 10 μl with water, the mixture was heated to 65° C. for 5minutes and then allowed to cool to room temperature. For the elongationreaction 2.5 μl of each of dTTP, dATP, dGTP and dCTP and 1 μl ATP (allat 10 μM) were added, followed by 1 μl (1 unit) of E. coli DNApolymerase (Klenow fragment) and 1 μl (1 unit) of T4 DNA ligase. Themixture was then incubated overnight at 14° C. and used to transform E.coli JM 109 (Yanisch-Perron et al. Gene 33, 103 (1985)) as described(Messing, supra).

To identify mutant clones by differential hybridization, plaques onnutrient agar were transferred to Gene Screen filters (New EnglandNuclear). The DNA was cross-linked to filters by irradiating them in aUV Stratalinker Model 1800 using the auto cross-link mode (Stratagene).They were then incubated for one hour in 6×SSC(0.9M NaCl/0.09MNa.citrate) containing 1% SDS at 60° C. For the hybridization, theoligonucleotide primer above (8 pmoles) was end-labeled with T4polynucleotide kinase and γ³²P-labeled ATP and incubated with thefilters overnight in 6×SSC, 0.5% SDS and 125 ug/ml herring sperm DNA.The hybridization temperatures were chosen according to estimates ofoligonucleotide melting points. Generally the hybridization temperaturewas approximately 10° C. less than the melting point. The next day, thefilters were washed two times with 6×SSC/1% SDS at hybridizationtemperature followed by two washes with 6×SSC at hybridizationtemperature and subjected to autoradiography. If necessary, the filterswere then washed with 6×SSC at increasing temperatures until little orno hybridization was detected to plaques having the wild-type MGDF cDNAsequence. Clones that gave positive hybridization signals under theseconditions were identified and retransfected into JM109 to isolate apure clone. Dideoxy chain termination sequence analysis indicated thatthe mutations were present.

Double stranded m13 MGDF 1-174 DNAs carrying the desired changes wererecovered from JM109 transfected cells with QIAGEN kits (ChatsworthCalif.) using methods supplied by the manufacturer. The DNAs weredigested with XbaI and SalI and the 605 bp MGDF DNA fragments wereisolated. pDSRα2 was digested with XbaI and SalI. The vector fragmentwas isolated and ligated to the MGDF fragments above. Recombinantplasmids were identified by restriction analysis. The resulting plasmids(designated MGDF 1-174-X where X is the analog number) contain DNAencoding MGDF analogs having altered amino acid residues at theindicated positions. The resultant plasmids were then sequenced again toconfirm the presence of the desired mutations.

An analog, MGDF 174-15 was constructed that had two additional N-linkedglycosylation sites at positions 30 and 120. PDSRα2MGDF 174-4 containingAsn30 and Thr32 mutations was digested with XbaI and PstI restrictionenzymes and the approximately 385 nt DNA fragment was isolated.PDSRα2MGDF 174-10 containing Asn120 and Thr122 mutations was digestedwith PstI and SalI restriction enzymes and the approximately 220 nt DNAfragment was isolated. pDSRα2 was digested with XbaI and SalI. Thevector fragment was isolated and ligated to the MGDF fragments above.This resulted in PDSRα2 MGDF 174-15 that contains Asn30, Thr32, Asn120and Thr122 substitutions.

These general procedures were used to construct the MGDF analogs shownin Table 1. The DNA sequence changes for each of the analogs are shown;otherwise the oligonucleotide primers used for mutagenesis had sequencescomplimentary to those of human MGDF.

TABLE 1 MGDF ANALOGS HAVING SITES FOR N-LINKED CARBOHYDRATE CHAINSAnalog/ Amino Acid Sequence Species No. Substitution Changes N1 NONENONE (amino acids 1-174 of FIG. 1) N2 Leu²²−>Asn²² CCT−>AAT N3Arg²⁵−>Asn²⁵ AGA−>AAC N4 Pro³⁰, Val³²−>Asn³⁰, Thr³² CCA, GTT−>AAC, ACCN5 Pro³⁸, Leu⁴⁰−>Asn³⁸, Thr⁴⁰ CCT, CTG−>AAT, ACG N6 Leu⁸⁶−>Asn⁸⁶CTC−>AAC N7 Gly⁸², Pro⁸³−>Asn⁸², Ala⁸³ GGA, CCC−>AAC, GCC N8 Ser⁸⁷,Leu⁸⁹−>Asn⁸⁷, Thr⁸⁹ TCA, CTC−>AAC, ACC N9 Glu⁹²−>Asn⁹² GAG−>AAC  N10Ala¹²⁰, Lys¹²²−>Asn¹²⁰, Thr¹²² GCT, AAG−>AAT, ACC  N11 Pro³⁶, Pro³⁸,Leu⁴⁰−> CCT, CCT, CTG−> Ser³⁶, Asn³⁸, Thr⁴⁰ TCT, AAT, ACG  N12Ser⁸⁸Leu⁹⁰−>Asn⁸⁸, Thr⁹⁰ TCC, CTG−>AAC, ACG  N13 Thr⁵³, Met⁵⁵−>Asn⁵³,Thr⁵⁵ ACC, ATG−>AAT, ACG  N14 Thr⁵⁸, Ala⁶⁰−>Asn⁵⁸, Thr⁶⁰ ACC, GCA−>AAC,ACA  N15 Pro³⁰, Val³², Ala¹²⁰, Lys¹²²−> CCA, GTT, GCT, Asn³⁰, Thr³²,Asn¹²⁰, Thr¹²² AAG−>AAC, ACC, AAT, ACC Note: Analogs N2-N15 aresynonymously referred to herein as analogs 2-15. Further, as usedherein, for example, [Asn²²] MGDF means that an asparagine has beensubstituted for the amino acid at position 22 in the particular MGDFspecies being considered, which is preferably a human sequence having atleast amino acids 7-151 of FIG. 1 (including the preferred human MGDFsequences set forth herein above). Thus, substitution of an asparagineresidue for a leucine residue at position 22 of MGDF 1-174 (humansequence) yields an MGDF analog that may be respresented by [Asn²²] MGDF1-174.

Plasmids designated pDSRα2 1-174-X (where X is the analog number) wereconstructed by inserting MGDF DNA into pDSRα2. The expression vectorpDSRα2 is generally described in WO 90/14363 (1990). pDSRα2MGDF 1-174-Xplasmids were made by digestion of pDSRα2 with XbaI and SalI. The vectorfragment was isolated and ligated to the approximately 605 bp fragmentscontaining the desired sequences.

EXAMPLE 5 Expression of MGDF and MGDF Analogs in COS Cells

cDNA clones of human MGDF and MGDF analogs described in Table 1 weretransferred into COS-1 cells (ATCC No. CRL-1650) by electroporation.COS-1 cells were harvested from semi-confluent dishes, washed withmedium (Dulbecco's modified essential medium containing 10% fetal bovineserum and 1% L-glutamine/penicillin/streptomycin (Irvine Scientific))and resuspended at 6×10⁶ cells/ml. One half ml of cells was transferredto a 0.2 cm electroporation cuvette (Bio-Rad) and electroporated with aBTX Electroporation System Electrocell Manipulator 600 at 650 uF and 130volts on the low voltage setting with 50 μg of plasmid DNA encoding theMGDF analog. The electroporated cells were plated on 100 mm tissueculture dish in 10 ml of medium. Twelve to twenty four hours afterplating the medium was replaced with 10 ml of fresh medium. Theconditioned medium was collected 3 to 5 days after electroporation.

EXAMPLE 6 Characterization of MGDF and MGDF Analogs

A. Determination of Carbohydrate Addition

A volume of supernatant containing approximately 30-60 ng MGDF or MGDFanalog from COS cells transfected with MGDF analog cDNAs as described inExample 5 was immunoprecipitated overnight at room temperature with arabbit anti-MGDF polyclonal antibody. In some cases where expression waslow, a maximum volume of approximately 8-9 ml was used forimmuno-precipitation. The antibody was raised to MGDF 1-163 that hadbeen expressed and purified from E. coli. Thirty μl of 1:1 ProteinA-Sepharose in phosphate buffered saline (PBS) containing 0.1% sodiumazide was added to the immunoprecipitate and allowed to incubate for onehour at room temperature. The samples were centrifuged, washed with PBSand resuspended in SDS sample Buffer (0.125 M Tris-HCl pH 6.8/4% SDS/20%glycerol/10% β-mercaptoethanol/0.001% bromophenol blue). The sampleswere analyzed by 12% SDS-polyacrylamide gel electrophoresis, transferredto nitrocellulose and subjected to Western analysis as described(Burnette et al., Anal. Biochem. 112:195-203 (1981); Elliott et al.,Gene 79:167-180 (1989)) using a mouse anti-MGDF monoclonal antibodyraised to a synthetic peptide corresponding to amino acid residues 1-31of FIG. 1. The MGDF containing bands were visualized using an ECL kit(Amersham).

FIG. 5 shows that COS cell supernatants from cells transfected withanalogs MGDF 174-4, MGDF 174-7 and MGDF 174-10 DNA revealed increasedsize compared to human sequence MGDF 174-1. FIG. 6 shows that COS cellsupernatants from cells transfected with MGDF 174-13, MGDF 174-14 andMGDF 174-4 DNA also had increased size compared to human sequence MGDF.This increased size is indicative of an additional N-linked carbohydratechain. MGDF analog 174-15 contains two additional N-linked glycosylationsites. FIG. 6 indicates that this analog has material with a sizegreater than analogs containing only 1 additional N-linkedglycosylation. The sizes of the proteins were estimated from theirmobility on SDS-PAGE relative to protein standards of known molecularweight. The estimated sizes of the larger bands calculated from FIG. 6is shown in Table 2. This result indicates that MGDF 174-15 contains 2additional N-linked chains. Western blot analyses of other selectedanalogs are also shown in FIG. 6.

TABLE 2 N-Linked Carbohydrate Estimates # of Potential MolecularN-Linked MGDF (1-174) Molecular Weight Shift (Da) Chains Analog Weight(Da) (Over Native) (@4 KDa/Site) N1 (Native) 23500 0 0 N4  28700 5200 1N7  27200 3700 1 N10 27200 3700 1 N13 26700 3200 1 N14 28700 5200 1 N1533500 10000 2

An experiment was performed to show that the increased size of MGDFanalogs is due to N-linked carbohydrate. COS cell conditioned mediumcontaining MGDF was immunoprecipitated and washed with PBS as describedabove. To each tube was then added 10 μl 0.5% SDS and each sample wasboiled for 3 minutes. Then the following components were added: 10.8 μlof 0.5M NaPO₄ pH 8.6, 5 μl of 7.5% nonidet P40 and 3 μl of 250 unit/mlN-glycanase (Genzyme). N-glycanase treatment removes N-linkedcarbohydrate. Samples were incubated for 6 hours at 37° C. The reactionwas stopped by the addition of SDS-PAGE sample buffer and then subjectedto SDS-PAGE Western analysis (12% acrylamide) using an anti-MGDFmonoclonal antibody and an anti-mouse ECL Western Detection Kit(Amersham) as described above. An analysis of N-linked chains using thismethod is shown in FIG. 7 for human MGDF and MGDF analogs. Followingtreatment with N-glycanase the mobility on Western blot for MGDF 174-4,MGDF 174-7 and MGDF 174-10 was reduced to that of MGDF 174-1. Asexpected, treatment of MGDF 174-1 with N-glycanase had no effect onmobility because MGDF 174-1 has no N-linked glycosylation sites. Theseresults indicate that the increased size observed is due to addition ofN-linked carbohydrate.

B. Analysis of O-linked carbohydrate on MGDF

To analyze the contribution of O-linked carbohydrate to human MGDF,various forms of the protein were purified from CHO cell conditionedmedia as described above. Each form received +/− treatment withO-glycanase (Glycopeptide alpha-N-acetylglactos-aminidase, OxfordGlycoSystems). O-glycanase removes O-linked carbohydrate fromglycoproteins. The E. coli expressed version of each form was used as anunglycosylated control. To resolve the difference in molecular weightcontributed by O-linked carbohydrate, it was necessary to remove anyN-linked carbohydrate first. Since the full length version, MGDF 1-332,contains N-linked carbohydrate, the CHO cell expressed full lengthsamples received N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) treatment as described above for COS cell expressedMGDF analogs, except that the N-glycanase treatment was an overnightincubation. Before proceeding with the O-glycanase treatment on fulllength (1-332) MGDF, the pH range of the sample was adjusted to pH6.0-pH 7.0 with 1/15 volume of 100 mM acetic acid, pH 2.2. One microgramof protein was denatured by boiling for 3 minutes in SDS and incubatedat 37° C. for 60 minutes with 1 U/ml neuraminidase (sialidase, fromArthrobacter urefaciens, Boehringer Mannheim) in 1 mM calcium acetate,pH 6.8 and 20 mM sodium phosphate, pH 6.8. Subsequent treatment withO-glycanase was done by adding 5 mU of enzyme in a final volume of 100ul, followed by an overnight incubation at 37° C. Proteins (0.2 ug/lane)were separated by SDS-PAGE (15% acrylamide). Following transfer to 0.2um nitrocellulose and overnight incubation with anti-MGDF polyclonalantibody the MGDF proteins were visualized using an anti-rabbit ECLWestern Detection Kit (Amersham). FIG. 3 shows a Western blot of fourdifferent forms of human MGDF. Full length MGDF 1-332 is represented inlanes 1-3, MGDF 1-174 lanes 4-6, MGDF 1-163 lanes 7-9, and MGDF 1-153lanes 10-12. Treatment with neuraminidase and O-glycanase, shown inlanes 2, 5, 8, and 11, reduced the molecular weight to that ofunglycosylated materials, lanes 3, 6, 9, and 12. In every case themobility increased to that of the unglycosylated version expressed in E.coli. These results indicate that the larger sized bands, in lanes 1, 4,7, and 10 are due to O-linked carbohydrate. The molecular weights ofeach of the bands was estimated by comparing their mobilities toproteins of known molecular weight. As seen in Table 3 which showsestimated molecular weights of the different proteins, the apparentshift in mobility could account for as many as 14 O-linked carbohydratechains (assuming 950 daltons/chain) for MGDF 1-332, 9 chains for MGDF1-174, 4 chains for MGDF 1-163, and 2 chains for MGDF 1-153. The samplerun in lane 2 is full length MGDF 1-332. It would appear that thisprotein was degraded, possibly due to extended incubation inglycoenzymes at 37° C. Therefore, the E. coli expressed unglycosylatedversion in lane 3 was used to calculate the approximate molecular weightof O-linked carbohydrate added to CHO cell expressed MGDF 1-332. Theseresults are consistant with the presence of carbohydrate on all the CHOexpressed forms of MGDF tested. The presence of O-linked carbohydratewas confirmed for CHO cell expressed MGDF 1-332, 1-174, and 1-163 bydirect analysis of monosaccharide composition. Sialic acids, GalNAc andGal were released from glycoproteins by acid hydrolysis. Themonosaccharides were detected by high pressure anion exchangechromatography and pulsed amperometric detection. All three sugars weredetected in each of the forms of MGDF. This result is indicative of thepresence of sialic acid containing O-linked carbohydrate. This datacorrelates with the in vivo data as seen in FIG. 4 where CHO cellexpressed forms of MGDF were all more active in vivo than the equivalentforms expressed in E. coli. Thus, the presence of carbohydrate enhancesthe in vivo activity of MGDF.

TABLE 3 O-Linked Carbohydrate Calculations O-Glycanase # of PotentialMGDF Treatment Molecular Molecular O-Linked Chains Form (+/−) Weight(Da) Weight Shift (@950 Da/Chain) 1-332 − 54200 13600  14 ″ E. coliversion 40600 1-174 − 24600 8600 9 ″ + 16000 1-163 − 18400 3900 4 ″ +14500 1-153 − 15200 2300 2 ″ + 12900

EXAMPLE 7 MGDF ELISA Assay

Polyclonal antibody production—New Zealand White rabbits werehyperimmunized over a period of three months with recombinant human MGDF1-163 produced in E. coli. Antisera from six rabbits exhibiting highantibody titers were pooled and specific anti-MGDF antibodies wereaffinity purified.

Affinity purification—Recombinant human MGDF 1-163 was covalentlyattached to Actigel-ALD (Sterogene Bioseparations, Inc.) according tothe manufacturer's instructions. An aliquot of the rabbit antisera poolwas added to the MGDF affinity gel, and the slurry was agitated gentlyon a rocker platform overnight a 4-8° C. Unbound serum proteins werewashed from the gel bed with PBS and specifically bound anti-MGDFantibodies were then eluted with ImmunoPure Gentle Ag/Ab Elution Buffer(Pierce Chemical Co.) Recovered antibodies were dialyzed against severalchanges of PBS, then the antibody solution was concentrated in an Amiconstirred cell ultrafiltration unit and the resultant antibody concentratewas the source of specific anti-MGDF antibodies subsequently used forwell coating and enzyme conjugate preparations.

ELISA reagents—Immulon 4 Removawell Strips (Dynatech Laboratories, Inc.)were coated with affinity purified rabbit anti-MGDF antibodies. Affinitypurified antibodies were diluted in 0.1 M sodium bicarbonate (freshlyprepared pH about 8.2) to a concentration of 2.5 ug/ml. Each wellreceived 100 ul of antibody and the plates were incubated for 24 hrs atroom temperature in a sealed and humidified chamber. Then, 200 ul of ablocking solution consisting of 1% fetal bovine serum 5% sucrose in TEN(50 mM Tris 7.4/10 mM EDTA/150 mM NaCl) was added to each well andplates were incubated and additional 24 hrs at room temperature in asealed and humidified chamber. Combined coating and blocking solutionswere removed from the wells. An additional overcoating/blocking step wasincluded: 300 ul of SuperBlock Blocking Buffer in PBS (Pierce ChemicalCo.) was added to each well. After standing at room temperature forabout 5 min. this solution was removed and the wells were allowed to airdry at room temperature for 24 hrs. The coated wells were stored insealed plastic bags at 4-8° C. until used in the MGDF ELISA.

Affinity purified anti-MGDF antibodies from a rabbit antisera pool werecovalently coupled to horseradish peroxidase (HRPO) for use as thesignal generating antibody. The affinity purified antibodies werederivatized with iminothiolane HCl (Fluka Chemical Corp.). Separately,HRPO was derivatized with N-succinimidyl 6-maleimidocaproate (FlukaChemical Corp.). The two activated proteins were combined to permitcovalent coupling. The reaction mixture was then chromatographed down aFPLC Superose 6 (Pharmacia) column to isolate the antibody:HRPOconjugate of the desired molecular weight (i.e. about 200 kD). Fractionscontaining the desired conjugate were combined and concentrated in aCentricon 30 (Amicon Division, W.R. Grace & Co.) and stored as a 50%glycerol solution at −20° C. This anti-MGDF Ab:HRPO concentrate wasdiluted into 2% fetal bovine serum in PBS for use in the ELISA. Thefinal concentration of conjugate used in the ELISA was 250-500 ng/ml.

Recombinant human MGDF 1-163 produced in E. coli cells, was used for thepreparation of standards. This MGDF was diluted into 2% fetal bovineserum (Sigma Chemical Co.) in TEN buffer containing 0.05% thimerosal asa preservative. Standards prepared contained 1.0, 0.5, 0.25, 0.125 and0.062 ng/ml MGDF.

Assay-100 ul of MGDF standards or samples was added to wells thenincubated for 18-24 hrs at room temperature in a sealed and humidifiedchamber. The well contents and residual solution were then removed andthe wells washed once with wash solution (0.05% Tween 20 in TEN buffer).Anti-MGDF Ab:HRPO conjugate solution (100 ul) was added to each well andthen incubated for 2 hrs at room temperature in a sealed and humidifiedchamber. The contents of wells were removed then washed 4 times with0.05% Tween 20 in TEN buffer. For color development, 100 ul ofTMB/peroxide substrate solution (Kirkegaard & Perry Solutions A & Bmixed 1:1) was added and incubated for 20 min at room temperature. Thereaction was stopped by addition of 100 ul stop solution (0.5 N sulfuricacid) and the absorbance was read at 450 nm on microtiter plate reader.Concentrations of MGDF in samples were calculated from a standard curvegenerated by using a curve fit program.

EXAMPLE 8 Biological Activity of MGDF 1-174 Analogs in a Short-TermLiquid Culture Megakaryocyte Assay

Analogs of MGDF(1-174) were prepared as described above and assayed fortheir ability to stimulate the growth of megakaryocytes in liquidculture. CD34 selected cells isolated from human leukapheresis units(Nichol et al., Stem Cells 12:494-505 (1994)) were plated at 2×10⁵/ml inculture medium (IMDM/1% Pen-Strep Glutamine/1% Non-essential AminoAcids/1% MEM Na-Pyruvate/1% MEM Vitamins/10% deionized BSA/10% normalhuman AB plasma/10 uM alpha-thiacylglycerol/20 ug/ml L-Asparagine). Inaddition, 1.5 ul of COS-1 conditioned medium containing MGDF(1-174) orMGDF(1-174) analog was added to each well. The final volume was 15 ul inTerasaki-style microtiter tissue culture plates (Vangard International).Cells were incubated at 37° C. for eight days in humidified boxes in 5%CO₂, fixed directly to the culture wells with 1% glutaraldehyde, andthen incubated with a monoclonal antibody cocktail consisting ofanti-GPIb, anti-GPIIb, (Biodesign) and anti-GPIb (Dako, Carpinteria,Calif.). The immune reaction was developed with astreptavidin-β-galactosidase detection system (HistoMark, Kirkegaard andPerry). Megakaryocytes, identified by the darker color (blue in actualphotographs), appear in FIG. 8.

Panels A and D of FIG. 8 are the positive and negative controlsrespectively. The well pictured in panel A received 37.5 pg of wild typeMGDF 1-174 COS-1 conditioned medium and shows substantial megakaryocytegrowth. Panel D received 1.5 ul of COS-1 mock conditioned medium andshows no growth. Panels B and C of FIG. 8 are MGDF 1-174 analogs 7 and10 respectively. Panel B received 9.0 pg of MGDF COS-1 conditionedmedium while panel C received 27 pg and both show excellentmegakaryocyte growth.

This experiment indicates that the analogs of MGDF tested are capable ofstimulating the growth of human megakaryocytes in vitro.

EXAMPLE 9 Biological Activity of MGDF 1-174 Analogs in an In Vitro CellProliferation Assay

Analogs of MGDF(1-174) were prepared as described above and assayed fortheir ability to stimulate the proliferation of 32D-MPL cells. Toconstruct 32D-MPL cells, the full length human Mpl receptor sequence(Vigon, I., et al., PNAS 89:5640-5644 (1992)) was subcloned into anexpression vector containing the transcriptional promoter of MoloneyMurine Sarcoma virus. Six ug of this construct and 6 ug of anamphotropic retroviral packaging construct (Landau, N. R., Littman, D.R., Journal of Virology 66:5110-5113 (1992)) were transfected into 3×10⁶293 cells using a CaPO₄ mammalian transfection kit (Stratagene). Thesame cells were retransfected after 2 days and again after 4 days. Theday after the last transfection the 293 cells were cocultivated with theIL-3 dependent murine cell line (32D, clone 23; Greenberger et al., PNAS80:2931-2936 (1983)). After 24 hours, the 32D cells were rescued andbanded in a BSA gradient (Path-o-cyte; Miles Inc.). Cells were expandedin 1 ng/ml murine IL-3 and then were selected for growth in 20% APK9serum (Bartley et al., Cell 77:1117-1124 (1994). Cells were sorted forcell surface expression of receptor by FACS using a polyclonal rabbitantipeptide (MPL) serum. These cytokine dependent murine 32D-MPL cellsare responsive to MGDF. 32D-MPL cells were grown in MEM mediumcontaining 10% Fetal Clone II Serum (Hyclone Laboratories) and 1.0 ng/mlmuIL3 to a cell density of 1×10⁶ cells/ml. Cells were collected bycentrifugation (approx. 500×G) and washed twice in growth medium lackingmuIL3 and resuspended at 1×10⁵ cells/ml. An extended twelve point MGDFstandard curve was prepared using rHuMGDF(1-163) and ranges from 5000 to1 pg/ml. A volume of 100 ul of each dilution of standard MGDF or assaysample was added to appropriate wells of a 96 well microtiter tissueculture plate containing 100 ul of resuspended cells (10,000 cells/well)and incubated in a humidified incubator at 37° C. and 10% CO₂. After 48hours, 40 ul of MTS reagent (Aqueous Non-Radioactive Cell ProliferationKit, Promega) was added to each well and 14-18 hours later the plateswere read on a plate reader at 490 nM. The in vitro activity in sampleswas calculated from a dose response curve for each sample. One unit wasdefined as the amount of MGDF in each sample required to give 50% ofmaximal stimulation. Specific activity was calculated by dividing thebiological activity in units/ml by the MGDF concentration in ng/ml asdetermined by MGDF ELISA.

The specific biological activity of MGDF analogs transfected andexpressed in COS cells is shown in Table 4. The effect of the amino acidsubstitutions on carbohydrate addition is also summarized. Purifiedhuman sequence MGDF has an in vitro activity that was 200-300 unit/ng asdetermined by the above-mentioned assays. It is apparent from Table 4that MGDF analogs containing additional N-linked carbohydrate areexpressed as well as native sequence MGDF even when they containadditional carbohydrate chains (as determined in Example 6, Section A)e.g. MGDF 174-4 and MGDF 174-10. Both of these analogs retained full invitro biological activity also. Therefore the MGDF analogs containingN-linked carbohydrate can be expressed normally in mammalian cells andthey can have normal or enhanced in vitro biological activity.

TABLE 4 In Vitro Specific Number Activity Activity of N- Elisa (units/(units/ MGDF linked chains (ng/ml) ml) ng) Form Sequence (a) (b) (c) (d)MOCK NONE 0 <0.08 <10 <125 174-1 Native NA 25 5375 215 174-1 Native 031.4 8800 280 174-1 Native 0 31.75 NA NA 174-2 N22 0 NA NA NA 174-3 N25NA 1.85 636 344 174-4 N30T32 1 38 8830 232 174-4 N30T32 1 24 NA NA 174-5N38T40 0 1.2 <10 <8 174-6 N86 0 0.44 <10 <22 174-7 N82A83 0 to 1 6 2660443 174-7 N82A83 0 to 1 4.7 3080 655 174-9 N92 0 10.5 1970 188 174-10N120T122 1 20.4 5943 291 174-10 N120T122 1 33.7 9690 288 174-11S36N38T40 NA <0.625 <10 <16 174-11 S36N38T40 0 1.3 <10 <8 174-13 N53T550 to 1 67 18000 269 174-14 N58T60 0 to 1 17.9 4850 271 174-15N30T32N120T122 0 to 2 26 6420 247 NOTES ^(a)The number of additionalN-linked chains was estimated based upon the mobility of the analogpolypeptides in SDS gels as described in Example 6. ^(b)Quantities ofMGDF analogs in CHO cell supernatants were determined by ELISA assay asdescribed in the Examples. ^(c)In vitro activity was determined bymeasuring stimulation of thymidine uptake in 32D cells dependent on MGDFfor growth. ^(d)Ratio of in vitro activity of MGDF analog as measured byproliferation assays to amount of MGDF analog measured by MGDF ELISA.N.A. Not available.

While the invention has been described in what is considered to be itspreferred embodiments, it is not to be limited to the disclosedembodiments, but on the contrary, is intended to cover variousmodifications and equivalents included within the spirit and scope ofthe appended claims, which scope is to be accorded the broadestinterpretation so as to encompass all such modifications andequivalents.

1. An analog of megakaryocyte growth and development factor (MGDF),wherein (a) said MGDF analog comprises a sequence of amino acidsselected from the group consisting of amino acid sequences 7-151 through1-332, inclusive, of SEQ ID NO: 1, (b) said analog of MGDF has at leastone added N-linked glycosylation site in said sequence of amino acids,(c) said MGDF analog has at least one added carbohydrate chain attachedto said added N-linked glycosylation site, and (d) said MGDF analog hasa biological activity of specifically stimulating or increasingmegakaryocytes or platelets.
 2. An analog according to claim 1, whereinsaid sequence of amino acids is selected from the group consisting ofMGDF 1-332 amino acids 1-332 of SEQ ID NO: 1 MGDF 1-191 amino acids1-191 of SEQ ID NO: 1 MGDF 1-183 amino acids 1-183 of SEQ ID NO: 1 MGDF1-174 amino acids 1-174 of SEQ ID NO: 1 MGDF 1-163 amino acids 1-163 ofSEQ ID NO: 1 MGDF 1-153 amino acids 1-153 of SEQ ID NO: 1 MGDF 1-152amino acids 1-152 of SEQ ID NO: 1 MGDF 1-151 amino acids 1-151 of SEQ IDNO: 1 MGDF 7-332 amino acids 7-332 of SEQ ID NO: 1 MGDF 7-191 aminoacids 7-191 of SEQ ID NO: 1 MGDF 7-183 amino acids 7-183 of SEQ ID NO: 1MGDF 7-174 amino acids 7-174 of SEQ ID NO: 1 MGDF 7-163 amino acids7-163 of SEQ ID NO: 1 MGDF 7-153 amino acids 7-153 of SEQ ID NO: 1 MGDF7-152 amino acids 7-152 of SEQ ID NO: 1 MGDF 7-151 amino acids 7-151 ofSEQ ID NO:
 1.


3. An analog of MGDF which is [Asn³⁰, Thr³²] MGDF.
 4. An analog of MGDFwhich is [Asn⁸², Ala⁸³] MGDF.
 5. An analog of MGDF which is [Asn¹²⁰,Thr¹²²] MGDF.
 6. An analog of MGDF which is [Asn⁵³, Thr⁵⁵] MGDF.
 7. Ananalog of MGDF which is [Asn⁵⁸, Thr⁶⁰] MGDF.
 8. An analog of MGDF whichis [Asn³⁰, Thr³², Asn¹²⁰, Thr¹²²] MGDF.
 9. A composition comprising atherapeutically effective amount of an MGDF analog according to any ofclaims 3 through 8, 1 and 2 together with a pharmaceutically acceptablediluent, adjuvant or carrier.